Effect of Microgravity on Fungistatic Activity of an α-Aminophosphonate Chitosan Derivative against Aspergillus niger

Biocontamination within the international space station is ever increasing mainly due to human activity. Control of microorganisms such as fungi and bacteria are important to maintain the well-being of the astronauts during long-term stay in space since the immune functions of astronauts are compromised under microgravity. For the first time control of the growth of an opportunistic pathogen, Aspergillus niger, under microgravity is studied in the presence of α-aminophosphonate chitosan. A low-shear modelled microgravity was used to mimic the conditions similar to space. The results indicated that the α-aminophosphonate chitosan inhibited the fungal growth significantly under microgravity. In addition, the inhibition mechanism of the modified chitosan was studied by UV-Visible spectroscopy and cyclic voltammetry. This work highlighted the role of a bio-based chitosan derivative to act as a disinfectant in space stations to remove fungal contaminants.     

Proposal from space radiation biologists. Importance of Centrifuge Facility in the study of biological effect by space radiation]

In microgravity, astronauts were constantly exposed to space radiation containing various kinds of radiation with a low-dose rate during long-term stays in space. It is very difficult to define the relative biological effectiveness (RBE) of space radiation under microgravity. In order to understand correct the RBE of space radiation, therefore, utilization of Centrifuge Facility is desired as a control experiment at orbit for removing other factors such as microgravity except space radiation. Here, we summarized the importance of Centrifuge Facility in the study of biological effect of space radiation.     

Changes of insulin in plasma and receptor for insulin in various tissues after the exposure of rats to space flights and hypokinesia

The explanation of the mechanism of the response to gravity changes is of great importance for the determination of the capacity of human subjects to adapt to the load of gravitational stress. Therefore several studies were performed to investigate the activity of endocrine system, since the hormones are involved in the regulation of physiological functions and metabolic processes. However the studies of endocrine system activity during altered gravity conditions, especially during the weightlessness are influenced by the several interventions in biomedical observations due to operational program of astronauts, wide variability in individual response and tolerance, use of extensive countermeasures, differences in the type of space missions and in the studies after landing also a hypergravity effect at landing and variability in postflight readaptation process. The significant changes of plasma insulin and glucose levels were observed in astronauts during space flights and in the first days of recovery period. In the first inflight period plasma insulin levels were increased, unchanged or decreased however after 4-5 weeks of exposure to weightlessness a decrease of insulin plasma levels were noted. After space flights an increase of plasma insulin levels were demonstrated in experimental animals and in human subjects. Since plasma insulin level is considered as most important factor involved in the regulation for insulin receptors in target tissues, an investigation of insulin receptors in various tissues was performed in rats exposed to space flight or to hypokinesia (model used for simulation of some effects of microgravity).     

Recording of blood pressure,heart rate and aortic nerve activity during parabolic flight in the rat via radio-telemetry

Exposure to microgravity induces cardiovascular deconditioning characterized by orthostatic hypotension when astronauts return to the earth. In order to understand the mechanism of cardiovascular deconditioning, it is necessary to clarify the changes in hemodynamics and the cardiovascular regulation system over the period of space flight. The telemetry system applied to freely moving animals will be a useful and appropriate technique for this kind of long term study of the cardiovascular system in the conscious animal during space flight. The purpose of the present study is twofold: firstly, to observe the detailed changes of arterial pressure and heart rate (HR) during microgravity elicited by the parabolic flight in order to study the acute effect of microgravity exposure on the cardiovascular system; and secondly, to test the feasibility of the telemetry system for recording blood pressure, HR and autonomic nervous activities continuously during space flight.     

Urinary albumin in space missions

Proteinuria was hypothesized for space mission but research data are missing. Urinary albumin, as index of proteinuria, was analyzed in frozen urine samples collected by astronauts during space missions onboard MIR station and on ground (control). Urinary albumin was measured by a double antibody radioimmunoassay. On average, 24h urinary albumin was 27.4% lower in space than on ground; the difference was statistically significant. Low urinary albumin excretion could be another effect of exposure to weightlessness (microgravity).     

Plasma hormone levels in human subject during stress loads in microgravity and at readaptation to Earth's gravity

In great part of the investigations of endocrine system functions in astronauts during space flights the plasma levels of hormones and metabolites were determined only in resting conditions, usually from one blood sample collection. Such levels reflected the psychical and physical state and new hormonal homeostasis of organism at the time of blood collection, however, the functional capacity of neuroendocrine system to respond to various stress stimuli during space flight remained unknown. The aim of present investigations was to study dynamic changes of hormone levels during the stress and metabolic loads (insulin induced hypoglycemia, physical exercise and oral glucose tolerance test) at the exposure of human subject to microgravity on the space station MIR. The responses of sympatico-adrenomedullary system to these stress and workloads were presented by Kvetnansky et al.     

Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station

The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.     

Suppression of antigen-specific lymphocyte activation in modeled microgravity

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.     

Analysis of possible causes of activation of gastric and the pancreatic excretory and incretory function after completion of space flight at the international space station

A comparative analysis of the excretory and incretory activity of the stomach and pancreas in astronauts soon after completion of space flights of various durations was performed. An increase in the fasting activity of gastric and pancreatic enzymes and hormones (insulin and C-peptide) in blood, reflecting the increased excretory and incretory activity of organs of the gastroduodenal region developing in microgravity, was demonstrated. The absence of subjects infected with Helicobacter pylori in the space flight crew excluded the involvement of this microorganism in the mechanism underlying the increase in the gastric secretory activity. The absence of correlation between the increase in the secretory activity of organs of the gastroduodenal region and the duration of the space flight allowed us to rule out the hypokinetic mechanism, which is associated with the duration of exposure to microgravity. It was concluded that the main mechanism underlying the changes in the functional state of the digestive system in space flight may be determined by the rearrangement of the venous hemodynamics of organs of the abdominal cavity, unrelated to the duration of exposure to microgravity. It was shown that, after completion of space flights and in ground-based experiments simulating the hemodynamic rearrangement occurring in microgravity, the increase in the basal excretory activity of gastroduodenal organs was not caused by gastrin secretion and occurred simultaneously with an increase in the secretion of insulin, which is considered as a putative hormonal component of the hemodynamic mechanism.     

The problem of artificial gravity: The current state and prospects

The review deals with the use of artificial gravity in manned space flights. The need for studying this problem is substantiated, with special emphasis on its implications for future interplanetary flights. The deconditioning of astronauts and a loss of their tolerance to gravitational loads despite the use of various preventive procedures are briefly discussed. The efficiency of artificial gravity generated by a short-arm centrifuge (SAC) is evaluated; the possibility of the use of an SAC in space flights (the effect of the main parameters of G-load on humans, and its tolerability, efficiency, etc.) is considered. Both Russian and foreign data are presented on the use of SAC for simulating microgravity effects under ground-based conditions (immersion and ANOH) and in experiments on board biosatellites. It is emphasized that all the data (both original and the data in the literature) testify to the efficiency of SAC as a preventive and therapeutic facility alleviating the negative effects of simulated microgravity. The problems that have not been resolved to date are also presented.     

A sustained hyper-g load as a tool to simulate space sickness

In April 1989 the three European scientist astronauts of the D1 Spacelab Mission were exposed to a 1.5 hours +3G centrifuge run in supine position, resulting in a linear acceleration along the subjects' x-axis. Afterwards, severe motion sickness symptoms were provoked by head movements (Sickness Induced by Centrifugation: SIC). The astronauts mentioned close similarities with what they experienced in space during the D1-Spacelab Mission in 1985, where head movements also provoked motion sickness symptoms (Space Adaptation Syndrome: SAS). Moreover, the astronauts agreed that the rank order of their susceptibility to SAS was the same as for SIC. It was therefore postulated that with this method SAS could be simulated on earth. Additionally, in otolith function tests following the centrifuge run, changes in visual-vestibular interaction were observed, which replicated objective findings obtained with the same astronauts immediately after the D1 Spacelab Mission. During the last couple of years a series of experiments has been carried out to determine the nature of the stimulus causing SIC, the incidence of SIC, and the underlying cardio-vascular and/or vestibular mechanisms. These experiments were carried out on several astronauts and some 50 'normal' healthy subjects. In the next sections the main findings of all these experiments and the implications are summarized.     

Impairment of antigen-specific cellular immune responses under simulated microgravity conditions

Summary Microgravity has been implicated to play a role in the observed immune dysfunction of astronauts and cosmonauts after either short-term or long-term space travel. These reports, together with studies describing increased levels of microorganisms in the space cabin environment suggest potential risk for in-flight incidences of infectious diseases. In order to understand the mechanism underlying these immune defects, it is important to have a ground-based model that would reliably mimic the effects of microgravity on antigen-specific immune function. We tested the utility of the rotating wall vessel (RWV) technology developed at NASA as a model system because in the RWV the culture medium and the cells rotate synchronously with the vessel, thereby creating simulated microgravity conditions. We compared the RWV to the conventional tissue culture flask (T-flask), for culturing immune precursor cells with cytotoxic T lymphocyte (CTL) activity against synthetic viral peptides. We observed a significant loss of antigen-specific CTL activity in RWV cultures, but not in those from the T-flask, irrespective of the peptide immunogen used for inducing the primary immune response in different mouse strains. Loss of CTL activity in RWV cultures coincided with a significant reduction in CD8⁺ cells as well as CD4⁺ cells and DEC205⁺ dendritic cells, suggesting adverse effects of RWV culturing on both the effector and accessory cells for the loss of antigen-specific CTL function. These results provide a strong parallel to the reported defects in cell-mediated immunity during space travel and strongly support the utility of the RWV technology as an effective ground-based model for identifying key steps in immune cell dysfunction related to microgravity.     

Circulatory and hormonal changes induced by microgravity

The absence (or decrease) of the hydrostatic pressure during space flights (microgravity state) or simulations of weightlessness (by immersion, bed rest or head-down tilt) result in a body fluid shift and an engorgement of the central circulation where mechanoreceptors involved in plasma volume regulation are located. Their activation induces the initial (first hours) hormonal response with a decrease in plasma vasopressin, renin and aldosterone and probably an increase in a natriuretic factor (Gauer reflex). Prolonged exposure to microgravity leads to more complex and often hypothetical responses: cardiovascular deconditioning, modifications in secretion and circadian rhythms of above cited hormones. After 24 years of studies on approximately 200 astronauts our knowledge of cardiovascular and hormonal adaptation to space flight is still at the beginning.     

Microgravity effect on testicular functions

In mammals spaceflight influences spermatogenesis since spermatogonial germ cell proliferation, compared to synchronous controls, is lightly decreased in irradiated or flown rats. Moreover, changes of the plasmatic testosterone production was described either in flight rats, or in rats maintained in simulated microgravity conditions. The hormonal levels of the astronauts change as it has been previously described, including hormones involved in the regulation of spermatogenesis such as testosterone and luteinizing hormone (LH). In microgravity conditions, human testosterone levels decreased whereas circulating LH levels increased. To study the effect of simulated microgravity on mammalian spermatogenesis we have utilized the Rotary Cell Culture System (RCCS) and we have cultured testicular fragments isolated from prepuberal rats in a chemically defined medium for three days under microgravity conditions. As control we have cultured the same amount of fragments at unit gravity. The morphology of the samples has been studied and the number of proliferating cells has been counted in control samples and in samples maintained in RCCS. The results indicate that the number of duplicating cells in the tubules was significantly increased in the microgravity-cultured fragments. The amount of testosterone secreted in the culture medium has been also evaluated and in RCCS samples the amount of the hormone was higher respect to the control samples.     

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions.     

Phenotypic characterization of Aspergillus niger and Candida albicans grown under simulated microgravity using a three-dimensional clinostat

The living and working environments of spacecraft become progressively contaminated by a number of microorganisms. A large number of microorganisms, including pathogenic microorganisms, some of which are fungi, have been found in the cabins of space stations. However, it is not known how the characteristics of microorganisms change in the space environment. To predict how a microgravity environment might affect fungi, and thus how their characteristics could change on board spacecraft, strains of the pathogenic fungi Aspergillus niger and Candida albicans were subjected to on-ground tests in a simulated microgravity environment produced by a three-dimensional (3D) clinostat. These fungi were incubated and cultured in a 3D clinostat in a simulated microgravity environment. No positive or negative differences in morphology, asexual reproductive capability, or susceptibility to antifungal agents were observed in cultures grown under simulated microgravity compared to those grown in normal earth gravity (1 G). These results strongly suggest that a microgravity environment, such as that on board spacecraft, allows growth of potentially pathogenic fungi that can contaminate the living environment for astronauts in spacecraft in the same way as they contaminate residential areas on earth. They also suggest that these organisms pose a similar risk of opportunistic infections or allergies in astronauts as they do in people with compromised immunity on the ground and that treatment of fungal infections in space could be the same as on earth.     

Proteomic Analysis of Mouse Hypothalamus under Simulated Microgravity

Exposure to altered microgravity during space travel induces changes in the brain and these are reflected in many of the physical behavior seen in the astronauts. The vulnerability of the brain to microgravity stress has been reviewed and reported. Identifying microgravity-induced changes in the brain proteome may aid in understanding the impact of the microgravity environment on brain function. In our previous study we have reported changes in specific proteins under simulated microgravity in the hippocampus using proteomics approach. In the present study the profiling of the hypothalamus region in the brain was studied as a step towards exploring the effect of microgravity in this region of the brain. Hypothalamus is the critical region in the brain that strictly controls the pituitary gland that in turn is responsible for the secretion of important hormones. Here we report a 2-dimensional gel electrophoretic analysis of the mouse hypothalamus in response to simulated microgravity. Lowered glutathione and differences in abundance expression of seven proteins were detected in the hypothalamus of mice exposed to microgravity. These changes included decreased superoxide dismutase-2 (SOD-2) and increased malate dehydrogenase and peroxiredoxin-6, reflecting reduction of the antioxidant system in the hypothalamus. Taken together the results reported here indicate that oxidative imbalance occurred in the hypothalamus in response to simulated microgravity.     

Leukocyte Activity Is Altered in a Ground Based Murine Model of Microgravity and Proton Radiation Exposure

Immune system adaptation during spaceflight is a concern in space medicine. Decreased circulating leukocytes observed during and after space flight infer suppressed immune responses and susceptibility to infection. The microgravity aspect of the space environment has been simulated on Earth to study adverse biological effects in astronauts. In this report, the hindlimb unloading (HU) model was employed to investigate the combined effects of solar particle event-like proton radiation and simulated microgravity on immune cell parameters including lymphocyte subtype populations and activity. Lymphocytes are a type of white blood cell critical for adaptive immune responses and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Mice were suspended prior to and after proton radiation exposure (2 Gy dose) and total leukocyte numbers and splenic lymphocyte functionality were evaluated on days 4 or 21 after combined HU and radiation exposure. Total white blood cell (WBC), lymphocyte, neutrophil, and monocyte counts are reduced by approximately 65%, 70%, 55%, and 70%, respectively, compared to the non-treated control group at 4 days after combined exposure. Splenic lymphocyte subpopulations are altered at both time points investigated. At 21 days post-exposure to combined HU and proton radiation, T cell activation and proliferation were assessed in isolated lymphocytes. Cell surface expression of the Early Activation Marker, CD69, is decreased by 30% in the combined treatment group, compared to the non-treated control group and cell proliferation was suppressed by approximately 50%, compared to the non-treated control group. These findings reveal that the combined stressors (HU and proton radiation exposure) result in decreased leukocyte numbers and function, which could contribute to immune system dysfunction in crew members. This investigation is one of the first to report on combined proton radiation and simulated microgravity effects on hematopoietic, specifically immune cells.